MicroRNA­24 alleviates colorectal cancer progression via a rs28382740 single nucleotide polymorphism in the long noncoding region of X­linked inhibitor of apoptosis protein.

Colorectal cancer (CRC) is one of the most prevalent malignant diseases worldwide. Recurrence is associated with the poor survival of patients with CRC. Targeted therapy and precision medicine for recurrent CRC may improve the clinical outcome. Therefore, finding biomarkers that can detect CRC early, assess its prognosis and survival, and predict its treatment response is key to improving the clinical prognosis. The aim of this study was to assess CRC recurrence by analyzing molecular differences using postoperative specimens. Whole-exome sequencing was first used to evaluate the molecular differences in CRC tissues from patients with recurrent disease, and the results were then verified with tissue array methods. The regulation of single nucleotide polymorphisms (SNPs) in long noncoding regions of interest was analyzed in the presence of target microRNAs (miRs) using luciferase assays. The results demonstrated that in patients with recurrent CRC, the G allele was mainly detected at the rs28382740 SNP in the 3'-untranslated region of the X-linked inhibitor of apoptosis (XIAP)-encoding gene. From the tissue arrays, 60% (3/5) of patients with the G allele of the rs28382740 SNP were diagnosed with CRC recurrence, whilst only 10% (1/10) of patients without the G allele had recurrent CRC (P=0.077). Furthermore, XIAP levels were high in non-CRC (50%; 2/4) and CRC (75%; 3/4) tissues of patients with recurrent disease and CRC (54.5%; 6/11) tissues of patients without recurrent disease. However, but only 9.1% (1/11) of non-CRC tissues of nonrecurrent patients had significantly high XIAP expression levels (P=0.022). Using a luciferase assay, it was demonstrated that miR-24s (miR-24-1-5p and miR-24-2-5p) targeting the rs28382740 SNP reduced XIAP levels in CRC cells with rs28382740 SNP genotype G. These results indicate that apoptosis-related proteins, such as XIAP, may be therapeutic targets or biomarkers for tumor development. The data from the present study support an inhibitory effect of miR-24s on XIAP expression. However, this inhibitory potency depends on the rs28382740 SNP genotype and may alleviate CRC progression by regulating the expression of XIAP.

MiR-24 regulates obstructive pulmonary disease in rats via S100A8.

PURPOSE: Chronic obstructive pulmonary disease (COPD) is a persistent inflammatory disorder characterized by minor airway inflammation and emphysema involving various cell types and cytokines. MicroRNAs (miRNAs) have emerged as critical regulators in the pathogenesis of lung diseases. This study investigates the impact of microRNA-24 (miR-24) on airway inflammatory responses in a rat model of COPD. MATERIALS AND METHODS: The model was established by combining cigarette smoke exposure and lipopolysaccharide stimulation, and rat lung tissues were transfected with adeno-associated viruses overexpressing miR-24. Pathological changes in the lung were assessed using hematoxylin and eosin staining. Levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha, interleukin-6, and interleukin-8, were measured using enzyme-linked immunosorbent assay. Expression of miR-24 and S100A8 was detected through quantitative reverse transcription PCR, while protein levels of S100A8, Toll-like receptor 4 (TLR4), and myeloid differentiation primary response 88 (MyD88) were assessed using western blotting. Bioinformatics analysis and dual-luciferase reporter assay were performed to determine the relationship between S100A8 and miR-24. RESULTS: The results demonstrated the downregulation of miR-24 in rats with COPD, and its overexpression resulted in a significant decrease in S1008 mRNA levels. Additionally, the protein level of S100A8 was significantly increased in the lung tissues of COPD rats. The upregulation of miR-24, however, not only inhibited the protein expression of S100A8, TLR4, and MyD88 in lung tissues but also reduced the release of pro-inflammatory cytokines in the plasma and bronchoalveolar lavage fluid, thereby attenuating inflammatory responses and pathological injuries in the lung. CONCLUSIONS: Our data suggest that miR-24 attenuates airway inflammatory responses in COPD by inhibiting the TLR4/MyD88 pathway via targeting S100A8.

MiR-24-3p modulates cardiac function in doxorubicin -induced heart failure via the Sp1/PI3K signaling pathway.

PURPOSE: The goal of this research was to explore the role of miR-24-3p in heart failure (HF), with a focus on its impact on the specificity protein 1 (Sp1)/phosphoinositide 3-kinase (PI3K) pathway. METHODS: HF rat and HF cell models were established using doxorubicin(Dox). Cardiac function was assessed through echocardiography, while histological changes were observed via hematoxylin-eosin (HE) staining. To further investigate the underlying mechanisms, HF cell models were treated with either an Sp1 inhibitor or a PI3K inhibitor. Additionally, models with miR-24-3p overexpression or silencing were constructed. N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were determined by ELISA. Cell apoptosis was evaluated using TUNEL staining, and lactate dehydrogenase (LDH) levels were measured by colorimetry. Reactive oxygen species (ROS) production was analyzed using flow cytometry. Related gene and protein expressions were assessed via qRT-PCR and Western blotting. Finally, the relationship between miR-24-3p and Sp1 was confirmed through dual-luciferase assays. RESULTS: Dox treatment increased the left ventricular internal diameter (LVIDd) while decreasing ejection fraction (EF) and fractional shortening (FS), leading to disorganized cardiomyocyte arrangement, cellular edema, and necrosis in rats. In HF rats, NT-proBNP, Caspase-3, and miR-24-3p expression levels were elevated, whereas Sp1 and PI3K mRNA and protein expression levels were decreased. Similarly, Dox-induced damage in H9c2 cardiomyocytes resulted in increased NT-proBNP, apoptosis, Caspase-3, LDH, ROS, and miR-24-3p expression, along with decreased Sp1 and PI3K expression. Treatment with either Sp1 or PI3K inhibitors exacerbated the Dox-induced cardiomyocyte damage, further elevating NT-proBNP, apoptosis, Caspase-3, LDH, ROS, and miR-24-3p expression levels. Notably, Sp1 inhibition reduced PI3K expression, and PI3K inhibition, in turn, suppressed Sp1 expression. Overexpression of miR-24-3p worsened Dox-induced cardiomyocyte damage, characterized by increased NT-proBNP, apoptosis, Caspase-3, LDH, and ROS expression, alongside reduced Sp1 and PI3K expression. In contrast, silencing miR-24-3p mitigated these detrimental effects and increased Sp1 and PI3K expression. Dual-luciferase assays confirmed that miR-24-3p directly targets Sp1. CONCLUSION: Dox induces cardiomyocyte damage, impairs cardiac function, and promotes cardiomyocyte apoptosis and oxidative stress. Silencing miR-24-3p offers a protective effect by activating the Sp1/PI3K signaling pathway in heart failure.

miR-24-3p inhibits lipid synthesis and progesterone secretion in chicken granulosa cells via ERK1/2 signaling pathway.

Normal follicular development is the basis for ovulation in poultry. Our previous sequencing analysis revealed a high expression of miR-24-3p in chicken follicles from degenerated ovaries, suggesting that miR-24-3p may modulate follicular development. Hence, this study investigated the specific mechanisms of miR-24-3p in regulating chicken follicular development. The results revealed that the proliferation, lipid synthesis, and progesterone secretion were significantly inhibited after miR-24-3p overexpression in chicken granulosa cells, vice versa by miR-24-3p knockdown. Dual-specificity phosphatase 16 (DUSP16) and thousand and one amino acid kinase 1 (TAOK1) were identified as potential target genes of miR-24-3p. Further validation revealed that knockdown of DUSP16 and TAOK1 suppressed proliferation, lipid synthesis, and progesterone secretion in chicken granulosa cells. Moreover, we observed that miR-24-3p, along with knockdown of DUSP16 and TAOK1, increased the phosphorylation levels of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Our previous study proved that activation of ERK1/2 inhibited lipid synthesis and progesterone secretion of chicken granulosa cells. In summary, we demonstrated that miR-24-3p targeting DUSP16 and TAOK1 disrupts lipid synthesis and progesterone secretion via ERK1/2 signaling pathway in chicken granulosa cells in vitro. These results may provide a new theoretical basis for resolving miRNAs regulation on reproductive performance of chickens.

Exosomal lncRNA HCP5 derived from human bone marrow mesenchymal stem cells improves chronic periodontitis by miR-24-3p/HO1/P38/ELK1 pathway.

Objective: The present study aimed to explore the function of human bone marrow mesenchymal stem cells (hBMMSCs)-derived exosomal long noncoding RNA histocompatibility leukocyte antigen complex P5 (HCP5) in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) to improve chronic periodontitis (CP). Methods: Exosomes were extracted from hBMMSCs. Alizarin red S staining was used to detect mineralised nodules. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to measure HCP5 and miR-24-3p expression. The mRNA and protein levels of alkaline phosphatase (ALP), osteocalcin, osterix, runt-related transcription factor 2, bone morphogenetic protein 2, osteopontin, fibronectin, collagen 1, heme oxygenase 1 (HO1), P38, and ETS transcription factor ELK1 (ELK1) were detected using RT-qPCR and Western blot. Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the HO1 and carbon monoxide concentrations. Heme, biliverdin, and Fe2+ levels were determined using detection kits. Micro-computed tomography, hematoxylin and eosin staining, ALP staining, tartrate-resistant acid phosphatase staining, ELISA, and RT-qPCR were conducted to evaluate the effect of HCP5 on CP mice. Dual luciferase, RNA immunoprecipitation, and RNA pulldown experiments were performed to identify the interactions among HCP5, miR-24-3p, and HO1. Results: The osteogenic ability of hPDLSCs significantly increased when co-cultured with hBMMSCs or hBMMSCs exosomes. Overexpression of HCP5 and HO1 in hBMMSCs exosomes promoted the osteogenic differentiation of hPDLSCs, and knockdown of HCP5 repressed the osteogenic differentiation of hPDLSCs. HCP5 knockdown enhanced the inflammatory response and repressed osteogenesis in CP mice. MiR-24-3p overexpression diminished the stimulatory effect of HCP5 on the osteogenic ability of hPDLSCs. Mechanistically, HCP5 acted as a sponge for miR-24-3p and regulated HO1 expression, and HO1 activated the P38/ELK1 pathway. Conclusion: HBMMSCs-derived exosomal HCP5 promotes the osteogenic differentiation of hPDLSCs and alleviates CP by regulating the miR-24-3p/HO1/P38/ELK1 signalling pathway.

Plasma EV-miRNAs as Potential Biomarkers of COVID-19 Vaccine Immune Response in Cancer Patients.

Cancer patients, prone to severe COVID-19, face immune challenges due to their disease and treatments. Identifying biomarkers, particularly extracellular vesicle (EV)-derived microRNAs (miRNAs), is vital for comprehending their response to COVID-19 vaccination. Therefore, this study aimed to investigate specific EV-miRNAs in the plasma of cancer patients under active treatment who received the COVID-19 booster vaccine. The selected miRNAs (EV-hsa-miR-7-5p, EV-hsa-miR-15b-5p, EV-hsa-miR-24-3p, EV-hsa-miR-145- 5p, and EV-hsa-miR-223-3p) are involved in regulating SARS-CoV-2 spike protein and cytokine release, making them potential biomarkers for vaccination response. The study involved 54 cancer patients. Plasma and serum samples were collected at pre-boost vaccination, and at 3 and 6 months post-boost vaccination. Anti-spike antibody levels were measured. Additionally, RNA was extracted from EVs isolated from plasma and the expression levels of miRNAs were assessed. The results showed a significantly positive antibody response after COVID-19 boost vaccination. The expression levels of EV-hsa-miR-7-5p, EV-hsa-miR-15b-5p, EV-hsa-miR-24-3p, and EV-hsa-miR-223-3p increased significantly after 6 months of COVID-19 booster vaccination. Interestingly, an increased expression of certain EV-hsa-miRNAs was positively correlated. Bioinformatic analysis revealed that these correlated miRNAs play a critical role in regulating the targets present in antiviral responses and cytokine production. These findings suggest that EV-hsa-miR-15b-5p, EV-hsa-miR-24-3p, and EV-hsa-miR-223-3p may be crucial in immune response induced by mRNA vaccines.

Involvement of oncomiRs miR-23, miR-24, and miR-27 in the regulation of alternative polyadenylation in glioblastoma via CFIm25 cleavage factor.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. The cleavage factor Im 25 (CFIm25), a crucial component of the CFIm complex, plays a key role in regulating the length of the mRNA 3'-UTR and has been implicated in various cancers, including GBM. This study sought to investigate the regulatory influence of specific microRNAs (miRNAs) on CFIm25 expression in GBM, a highly aggressive brain tumor. Bioinformatics analysis identified miRNA candidates targeting CFIm25 mRNA, and gene expression profiles from the NCBI database (GSE90603) were used for further analysis. Expression levels of CFIm25 and selected miRNAs were assessed using qRT-PCR in GBM clinical samples (n = 20) and non-malignant brain tissues (n = 5). Additionally, the MTT assay was performed to examine the effect of miRNA overexpression on U251 cell viability. Lentivectors expressing the identified miRNAs were employed to experimentally validate their regulatory role on CFIm25 in U251 cell lines, and Western blot analysis was conducted to determine CFIm25 protein levels. We observed significantly increased levels of miR-23, miR-24, and miR-27 expression, associated with a marked reduction in CFIm25 expression in GBM samples compared to non-malignant brain tissues. In particular, overexpression of miR-23, miR-24, and miR-27 in U251 cells resulted in CFIm25 downregulation at both the mRNA and protein levels, while their inhibition increased CFIm25 and reduced cell proliferation. These observations strongly implicate miR-23, miR-24, and miR-27 in regulating CFIm25 expression in GBM, emphasizing their potential as promising therapeutic targets for enhancing treatment responses in glioblastoma.

Biomarkers for Oral Squamous Cell Carcinoma (miR-24, miR-200, and miR-34): Screening and Detection MicroRNA.

INTRODUCTION: Oral squamous cell carcinoma (OSCC) includes about 90% of all oral malignant tumors, and most of them are diagnosed in advanced stages. This study investigated the expression changes of miR-24, miR-200, and miR-34 in saliva samples of patients with oral squamous cell carcinoma, for early diagnosis. METHODS: In this study, 30 patients and 30 healthy individuals were selected. After RNA extraction and cDNA synthesis, the expression levels of miR-24, miR-200, and miR-34 in saliva samples were measured and evaluated using the Real-Time PCR technique. RESULTS: Folding change calculation using 2^(-∆∆ Ct) refers to the relative difference in the expression of the markers of the two groups. The expression level of two biomarkers, miR-200 and miR-34, is decreased in patients compared to healthy people; and the expression level of miR-24 is increased in patients compared to healthy people. CONCLUSION: In general, considering the availability and convenience of saliva sample collection for early detection of the disease, this research result can be considered a diagnostic screening test. To further prove the research results, conducting more extensive studies with more samples is recommended.

Modulation of Serotonin-Related Genes by Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 in the Interleukin-1ß-Induced Inflammation Model of Intestinal Epithelial Cells.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition involving dysregulated immune responses and imbalances in the gut microbiota in genetically susceptible individuals. Current therapies for IBD often have significant side-effects and limited success, prompting the search for novel therapeutic strategies. Microbiome-based approaches aim to restore the gut microbiota balance towards anti-inflammatory and mucosa-healing profiles. Extracellular vesicles (EVs) from beneficial gut microbes are emerging as potential postbiotics. Serotonin plays a crucial role in intestinal homeostasis, and its dysregulation is associated with IBD severity. Our study investigated the impact of EVs from the probiotic Nissle 1917 (EcN) and commensal E. coli on intestinal serotonin metabolism under inflammatory conditions using an IL-1ß-induced inflammation model in Caco-2 cells. We found strain-specific effects. Specifically, EcN EVs reduced free serotonin levels by upregulating SERT expression through the downregulation of miR-24, miR-200a, TLR4, and NOD1. Additionally, EcN EVs mitigated IL-1ß-induced changes in tight junction proteins and oxidative stress markers. These findings underscore the potential of postbiotic interventions as a therapeutic approach for IBD and related pathologies, with EcN EVs exhibiting promise in modulating serotonin metabolism and preserving intestinal barrier integrity. This study is the first to demonstrate the regulation of miR-24 and miR-200a by probiotic-derived EVs.

A cross talk study on sitagliptin mediated reclamation on TGF ß signalling, DPP 4, miR-29a and miR-24 expression in PCOS rats fed with high fat-high fructose diet.

Polycystic Ovary Syndrome (PCOS) is a multifactorial reproductive, endocrine and metabolic disturbance which is very commonly observed in females of reproductive age group. The disease is still incurable however the use of synthetic drugs in combination with lifestyle is recommended. Accordingly, the present study was conducted to investigate the possible beneficial effects of sitagliptin on PCOS induced rats on control diet (CD)/high fat- high fructose diet (HFFD). PCOS was induced by giving testosterone propionate (TP) for 28 days to both the CD/HFFD rats and treated with STG i.p. for last 15 days. At the end of the experiment lipid profile, inflammatory markers, expression of NF-κB-p65, miR-24 and miR-29a, fibrotic and apoptotic proteins from ovary tissue were examined. Moreover, lipid accumulation and fibrosis of ovary tissue was further confirmed using Sudan III and Masson's trichrome stain. STG treated rats exerted a significant decrease in levels of cholesterol, TG, LDL-C, VLDL-C, IL-6 and TNF-α and increased HDL-C level, miR-24 and miR-29a expression. STG treated groups expressed significantly decreased expression of NF-κB-p65, TGF-ß1, p-Smad 2 and p-Smad 3 followed by no significant changes in the expression of BAX, caspase-9, caspase-3 and Bcl-2 in all the PCOS induced groups. Among all the CD/ HFFD fed groups, rats on HFFD showed more devastating effect which suggests that diet plays a major role in genesis of PCOS. In conclusion, current results reflect the potential impact of STG against dyslipidaemia, inflammation and fibrosis in PCOS rats via regulating dyslipidaemia and fibrosis via DPP 4 mediated miR-29a expression.

MiR-24-3p enhances the Treg/Th17 balance to improve cerebral ischemic injury by suppressing acetyl-CoA carboxylase 1 expression.

BACKGROUND: Targeting ACC1 (acetyl coenzyme A carboxylase 1) to restore the balance between T-helper 17 (Th17) cells and regulatory T cells (Tregs) through metabolic reprogramming has emerged as a promising strategy for reducing neuroinflammation following stroke. We examined the roles of potential miRNAs in regulating ACC1 expression in Tregs and treating ischemic stroke. METHODS: The expression of miR-24-3p in CD4+T cells of mice was confirmed. Then the protective effects of Ago-24-3p in a mouse model of prolonged occlusion of the distal middle cerebral artery (dMCAO) were examined. We analyzed the infiltration of Tregs and CD3+T cells into the brain and evaluated the improvement of neurological deficits induced by Ago-24-3p using the Modified Garcia Score and foot fault testing. RESULTS: Our investigation revealed that miR-24-3p specifically targets ACC1. Elevated levels of miR-24-3p have been demonstrated to increase the population of Tregs and enhance their proliferation and suppressive capabilities. Conversely, targeted reduction of ACC1 in CD4+T cells has been shown to counteract the improved functionality of Tregs induced by miR-24-3p. In a murine model of dMCAO, administration of Ago-24-3p resulted in a substantial reduction in the size of the infarct within the ischemic brain area. This effect was accompanied by an upregulation of Tregs and a downregulation of CD3+T cells in the ischemic brain region. In ACC1 conditional knockout mice, the ability of Ago-24-3p to enhance infiltrating Treg cells and diminish CD3+T cells in the ischemic brain area has been negated. Furthermore, its capacity to reduce infarct volume has been reversed. Furthermore, we demonstrated that Ago-24-3p sustained improvement in post-stroke neurological deficits for up to 4 weeks after the MCAO procedure. CONCLUSIONS: MiR-24-3p shows promise in the potential to reduce ACC1 expression, enhance the immunosuppressive activity of Tregs, and alleviate injuries caused by ischemic stroke. These discoveries imply that miR-24-3p could be a valuable therapeutic option for treating ischemic stroke.

Long noncoding RNA UNC5B-AS1 suppresses cell proliferation by sponging miR-24-3p in glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary CNS tumor, characterized by high mortality and heterogeneity. However, the related lncRNA signatures and their target microRNA (miRNA) for GBM are still mostly unknown. Therefore, it is critical that we discover lncRNA markers in GBM and their biological activities. MATERIALS AND METHODS: GBM-related RNA-seq data were obtained from the Cancer Genome Atlas (TCGA) database. The "edger" R package was used for differently expressed lncRNAs (DELs) identification. Then, we forecasted prospective miRNAs that might bind to lncRNAs by Cytoscape software. Survival analysis of those miRNAs was examined by the starBase database, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the miRNAs' target genes was conducted by the Gene Set Enrichment Analysis (GSEA) database and R software. Moreover, the proliferative ability of unc-5 netrin receptor B antisense RNA 1 (UNC5B-AS1) cells was evaluated by Cell Counting Kit-8 (CCK-8) analysis. Mechanistically, the regulatory interaction between UNC5B-AS1 and miRNA in GBM biological processes was studied using CCK-8 analysis. RESULTS: Our results indicated that overexpression of UNC5B-AS1 has been shown to suppress GBM cell growth. Mechanistically, miR-24-3p in GBM was able to alleviate the anti-oncogenic effects of UNC5B-AS1 on cell proliferation. CONCLUSION: The discovery of the novel UNC5B-AS1-miR-24-3p network suggests possible lncRNA and miRNA roles in the development of GBM, which may have significant ramifications for the analysis of clinical prognosis and the development of GBM medications.

Regulation of myo-miR-24-3p on the Myogenesis and Fiber Type Transformation of Skeletal Muscle.

Skeletal muscle plays critical roles in providing a protein source and contributing to meat production. It is well known that microRNAs (miRNAs) exert important effects on various biological processes in muscle, including cell fate determination, muscle fiber morphology, and structure development. However, the role of miRNA in skeletal muscle development remains incompletely understood. In this study, we observed a critical miRNA, miR-24-3p, which exhibited higher expression levels in Tongcheng (obese-type) pigs compared to Landrace (lean-type) pigs. Furthermore, we found that miR-24-3p was highly expressed in the dorsal muscle of pigs and the quadriceps muscle of mice. Functionally, miR-24-3p was found to inhibit proliferation and promote differentiation in muscle cells. Additionally, miR-24-3p was shown to facilitate the conversion of slow muscle fibers to fast muscle fibers and influence the expression of GLUT4, a glucose transporter. Moreover, in a mouse model of skeletal muscle injury, we demonstrated that overexpression of miR-24-3p promoted rapid myogenesis and contributed to skeletal muscle regeneration. Furthermore, miR-24-3p was found to regulate the expression of target genes, including Nek4, Pim1, Nlk, Pskh1, and Mapk14. Collectively, our findings provide evidence that miR-24-3p plays a regulatory role in myogenesis and fiber type conversion. These findings contribute to our understanding of human muscle health and have implications for improving meat production traits in livestock.

Danhong formula alleviates endothelial dysfunction and reduces blood pressure in hypertension by regulating MicroRNA 24 - Phosphatidylinositol 3-Kinase-Serine/Threonine Kinase- Endothelial Nitric Oxide Synthase axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Essential hypertension (EH) is one of the important risk factors of cardio-cerebrovascular diseases, and it can significantly increase the incidence and mortality of acute myocardial infarction, cerebral infarction and hemorrhage. Danhong Formula (DHF) was consisting of Radix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Honghua in Chinese) (Plant names have been checked with http://www.the plant list.org on June 28th, 2023) was approved by State Food and Drug Administration of China, that has been used for thousands of years in the treatment of cardiovascular diseases in China with proven safety and efficacy. Though our previous studies have found that DHF improved endothelial dysfunction (ED) and decreased high blood pressure (BP), the underlying mechanisms of its antihypertensive effect still remain unclear. AIM OF THE STUDY: This study investigated whether DHF regulated MicroRNA 24- Phosphatidylinositol 3-Kinase-Serine/Threonine Kinase- Endothelial Nitric Oxide Synthase (miR-24 - PI3K/AKT/eNOS) axis to produce antihypertensive effect and improve endothelial dysfunction. MATERIALS AND METHODS: Firstly, the chemical components of DHF were analyzed by UHPLC-MS. After that, BP was continuously monitored within the 1st, 3rd, and 4th week in SHR to evaluate the antihypertensive effect of DHF intraperitoneal injection. In addition, not only the contents of serum nitric oxide (NO), prostacyclin (PGI2), and angiotensin II (Ang II) were detected, but also the isolated aorta ring experiment was conducted to evaluate the vasomotoricity to evaluate of DHF on improving endothelial dysfunction. Key proteins or mRNA expression associated with miR-24 - PI3K/AKT/eNOS axis in aorta were detected by capillary Western blot, immunohistochemistry or RT-PCR to explore the underlying mechanisms. Index of NO, Ang II PGI2 and key proteins or mRNA expression were also conducted in miR-24-3p over-expression HUVECs model. RESULTS: Compared with SHR control group, DHF (4 mL/kg/day, 2 mL/kg/day, 1 mL/kg/day) treatment significantly reduced high BP in SHR and selectively increased acetylcholine (Ach) induced vasodilation, but not sodium nitroprusside (SNP) in a manner of concentration dependency in isolated aorta ring. DHF (4 mL/kg/day, 1 mL/kg/day) treatment was accompanying an increment of NO and PGI2, and lowering AngII in SHR. Moreover, DHF treatment significantly up-regulated expression of p-PI3K, p-AKT, mTOR, eNOS and p-eNOS, but down-regulated miR-24-3p expression in aorta. Compared with miR-24-3p over-expression HUVECs model group, DHF treatment inhibited miR- 24-3p expression and up-regulated p-PI3K, p-AKT, mTOR and eNOS mRNA expression. Similarly, DHF treatment increased PI3K, AKT, mTOR and eNOS protein expression in HUVECs by Western blot. CONCLUSIONS: These findings suggest that DHF alleviates endothelial dysfunction and reduces high BP in SHR mediated by down-regulating miR-24 via ultimately facilitating up-regulation of PI3K/AKT/eNOS axis. This current study firstly demonstrates a potential direction for antihypertensive mechanism of DHF from microRNA aspect and will promote its clinical applications.

Knockdown of miR-24 Suppressed the Tumor Growth of Cervical Carcinoma Through Regulating PTEN/PI3K/AKT Signaling Pathway.

Cervical cancer (CC) is the most prevalent malignant tumor in gynecology. Despite routine surgery, advanced CC is hard to remove completely. MicroRNA-24 (miR-24) regulates several types of tumors, but its regulatory function in CC was previously unknown. We established stable knockdown of miR-24 and phosphatase and tensin homolog (PTEN) in CC cells. We measured mRNA and protein expression with RT-PCR and western blotting. We evaluated cell proliferation, invasion, migration, and apoptosis with CCK8, Transwell, wound healing, and flow cytometry, respectively. We also examined the influence of miR-24 and PTEN on tumor growth in a metastatic tumor model in nude mice. The expression of miR-24 was significantly increased in CC tissues and cell lines (C-33A, HeLa S3, SiHa). MiR-24 inhibitor greatly suppressed PTEN/PI3K/AKT, while miR-24 mimic markedly activated this signaling pathway. Knockdown of PTEN significantly reversed the effects of miR-24 inhibitor on cell proliferation, invasion, migration, and apoptosis of CC cells. The significant inhibition effect of tumor growth and ki67 expression caused by miR-24 inhibitor was reversed by si-PTEN. MiR-24 inhibitor significantly suppressed cell proliferation, invasion, migration, epithelial-mesenchymal transition (EMT) process, and tumor growth, while promoting cell apoptosis. However, the influence of miR-24 inhibitor was markedly reversed by si-PTEN. Targeting miR-24 could provide a novel therapeutic strategy for the prevention and treatment of CC.

SHED-derived exosomes attenuate trigeminal neuralgia after CCI of the infraorbital nerve in mice via the miR-24-3p/IL-1R1/p-p38 MAPK pathway.

BACKGROUND: Microglial activation in the spinal trigeminal nucleus (STN) plays a crucial role in the development of trigeminal neuralgia (TN). The involvement of adenosine monophosphate-activated protein kinase (AMPK) and N-methyl-D-aspartate receptor 1 (NMDAR1, NR1) in TN has been established. Initial evidence suggests that stem cells from human exfoliated deciduous teeth (SHED) have a potential therapeutic effect in attenuating TN. In this study, we propose that SHED-derived exosomes (SHED-Exos) may alleviate TN by inhibiting microglial activation. This study sought to assess the curative effect of SHED-Exos administrated through the tail vein on a unilateral infraorbital nerve chronic constriction injury (CCI-ION) model in mice to reveal the role of SHED-Exos in TN and further clarify the potential mechanism. RESULTS: Animals subjected to CCI-ION were administered SHED-Exos extracted by differential ultracentrifugation. SHED-Exos significantly alleviated TN in CCI mice (increasing the mechanical threshold and reducing p-NR1) and suppressed microglial activation (indicated by the levels of TNF-α, IL-1ß and IBA-1, as well as p-AMPK) in vivo and in vitro. Notably, SHED-Exos worked in a concentration dependent manner. Mechanistically, miR-24-3p-upregulated SHED-Exos exerted a more significant effect, while miR-24-3p-inhibited SHED-Exos had a weakened effect. Bioinformatics analysis and luciferase reporter assays were utilized for target gene prediction and verification between miR-24-3p and IL1R1. Moreover, miR-24-3p targeted the IL1R1/p-p38 MAPK pathway in microglia was increased in CCI mice, and participated in microglial activation in the STN. CONCLUSIONS: miR-24-3p-encapsulated SHED-Exos attenuated TN by suppressing microglial activation in the STN of CCI mice. Mechanistically, miR-24-3p blocked p-p38 MAPK signaling by targeting IL1R1. Theoretically, targeted delivery of miR-24-3p may offer a potential strategy for TN.

Circ_0020014 mediates CTSB expression and participates in IL-1ß-prompted chondrocyte injury via interacting with miR-24-3p.

BACKGROUND: Recent studies have shown that circRNAs are involved in the pathogenesis of osteoarthritis (OA) by affecting various fundamental cellular characteristics of chondrocytes. The purpose of this paper is to investigate the role and regulatory mechanism of hsa_circ_0020014 (circ_0020014) in chondrocytes of OA. METHODS: The inflammatory cytokine interleukin 1 beta (IL-1ß) was used to stimulate human chondrocytes. Cell viability, proliferation, and apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 5-Ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays. Several protein levels were determined by western blotting (WB). Levels of inflammatory cytokines and malondialdehyde (MDA) were determined by enzyme-linked immunosorbent assay (ELISA). Relative expression of circ_0020014 was estimated by real-time polymerase quantitative chain reaction (RT-qPCR). Bioinformatics prediction combined with dual-luciferase reporter, RIP and RNA pull-down assays were done to probe into the regulatory mechanism of circ_0020014. RESULTS: Circ_0020014 was overexpressed in OA patient-derived articular cartilages and IL-1ß-stimulated chondrocytes. Silencing of circ_0020014 lighted IL-1ß-prompted chondrocyte proliferation repression, apoptosis, inflammation, and oxidative stress. Mechanically, circ_0020014 functioned as a miR-24-3p molecular sponge to regulate cathepsin B (CTSB) expression. Furthermore, miR-24-3p inhibition alleviated circ_0020014 knockdown-mediation repression of IL-1ß-urged chondrocyte injury. In addition, CTSB overexpression whittled miR-24-3p upregulation-mediated suppression of IL-1ß-urged chondrocyte injury. CONCLUSION: Our findings demonstrated that the circ_0020014/miR-24-3p/CTSB axis was associated with IL-1ß-prompted chondrocyte injury, supporting the involvement of circ_0020014 in the OA pathogenesis.

Wolf-Hirschhorn syndrome candidate 1 (Whsc1) methyltransferase signals via a Pitx2-miR-23/24 axis to effect tooth development.

Wolf-Hirschhorn syndrome (WHS) is a developmental disorder attributed to a partial deletion on the short arm of chromosome 4. WHS patients suffer from oral manifestations including cleft lip and palate, hypodontia, and taurodontism. WHS candidate 1 (WHSC1) gene is a H3K36-specific methyltransferase that is deleted in every reported case of WHS. Mutation in this gene also results in tooth anomalies in patients. However, the correlation between genetic abnormalities and the tooth anomalies has remained controversial. In our study, we aimed to clarify the role of WHSC1 in tooth development. We profiled the Whsc1 expression pattern during mouse incisor and molar development by immunofluorescence staining and found Whsc1 expression is reduced as tooth development proceeds. Using real-time quantitative reverse transcription PCR, Western blot, chromatin immunoprecipitation, and luciferase assays, we determined that Whsc1 and Pitx2, the initial transcription factor involved in tooth development, positively and reciprocally regulate each other through their gene promoters. miRNAs are known to regulate gene expression posttranscriptionally during development. We previously reported miR-23a/b and miR-24-1/2 were highly expressed in the mature tooth germ. Interestingly, we demonstrate here that these two miRs directly target Whsc1 and repress its expression. Additionally, this miR cluster is also negatively regulated by Pitx2. We show the expression of these two miRs and Whsc1 are inversely correlated during mouse mandibular development. Taken together, our results provide new insights into the potential role of Whsc1 in regulating tooth development and a possible molecular mechanism underlying the dental defects in WHS.

Invasive Breast Cancer: miR-24-2 Targets Genes Associated with Survival and Sensitizes MDA-MB-231 Cells to Berberine.

MicroRNA aberrations including that of miR-24-2 have been reported in various cancers. However, the target genes for miR-24-2 are yet to be identified and validated in invasive breast cancer and the triple-negative breast cancer (TNBC). Using in silico approaches and gene expression analyses, we identified and validated the target genes of miR-24-2 in invasive breast cancer, majority of which were TNBC. We studied the translational potential of these target genes using berberine in a TNBC cell line. Differentially expressed genes targeted by miR-24-2 were identified and analyzed for their survival effects using the The Cancer Genome Atlas-Breast Invasive Carcinoma (-BRCA) samples. Furthermore, we carried out protein-protein interaction, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, gene expression, and Kaplan-Meier survival analyses using common targets of miR-24-2 in invasive breast cancer/TNBC. We identified 11 biomarker candidate genes as crucial targets of miR-24-2. The survival of breast cancer patients was significantly associated with the low expressions of nine genes, including RACGAP1, KIAA1199, TIMM17A, LYRM7, IL1R1, SLC1A3, DTX4, L1CAM, and SAP30-like (SAP30L), and high expressions of two genes, SOD2 and HLA-DQB2. These in silico findings were validated by overexpressing miR-24-2 and assessing the expression pattern of these target genes in the TNBC MDA-MB-231 cells. miR-24-2 overexpression inhibited (by 20%; p < 0.001) cell proliferation and sensitized the anticancer effect of berberine. In all, this study reports on the novel target genes of miR-24-2 in invasive breast cancer/TNBC, and that miR-24-2 sensitizes MDA-MB-231 cells to berberine. These data lend evidence for the translational potentials of miR-24-2 for invasive breast cancer diagnostic and therapeutic innovation.

Acute myeloid leukemia-derived exosomes deliver miR-24-3p to hinder the T-cell immune response through DENN/MADD targeting in the NF-κB signaling pathways.

BACKGROUND: microRNAs (miRNAs) are known as potent gene expression regulators, and several studies have revealed the prognostic value of miRNAs in acute myeloid leukemia (AML) patient survival. Recently, strong evidence has indicated that miRNAs can be transported by exosomes (EXOs) from cancer cells to recipient immune microenvironment (IME) cells. RESULTS: We found that AML blast-released EXOs enhance CD3 T-cell apoptosis in both CD4 and CD8 T cells. We hypothesized that miRNAs present in EXOs are key players in mediating the changes observed in AML T-cell survival. We found that miR-24-3p, a commonly overexpressed miRNA in AML, was present in released EXOs, suggesting that EXO-miR-24-3p was linked to the increased miR-24-3p levels detected in isolated AML T cells. These results were corroborated by ex vivo-generated miR-24-3p-enriched EXOs, which showed that miR-24-3p-EXOs increased apoptosis and miR-24-3p levels in T cells. We also demonstrated that overexpression of miR-24-3p increased T-cell apoptosis and affected T-cell proliferation by directly targeting DENN/MADD expression and indirectly altering the NF-κB, p-JAK/STAT, and p-ERK signaling pathways but promoting regulatory T-cell (Treg) development. CONCLUSIONS: These results highlight a mechanism through which AML blasts indirectly impede T-cell function via transferred exosomal miR-24-3p. In conclusion, by characterizing the signaling network regulated by individual miRNAs in the leukemic IME, we aimed to discover new nonleukemic immune targets to rescue the potent antitumor function of T cells against AML blasts. Video Abstract.